Rafnweinstein3188

2-10) in the radiation group treated with RTA-408. Vascular density was preserved in the treatment group as compared to the radiated control. Nine rats were included in the second experiment, and transcriptome analyses in the treatment group revealed robust activation of antioxidant pathways with induced expression of genes associated with hypoxia and adipogenesis/angiogenesis.

Administration of RTA-408 during radiation treatment in a rat model resulted in transcriptome changes which appear to mitigate the toxic effects of radiation, preserving capillary networks and improving flap survival and tissue healing after subsequent surgery.

Foundational Evidence, Animal Research Laryngoscope, 2021.

Foundational Evidence, Animal Research Laryngoscope, 2021.Ectosymbionts, such as bacteria and parasites, are found on the surface of organisms throughout the world's ecosystems. Hosts have evolved a variety of mechanisms across ecosystems to rid themselves of ectoparasites or other skin irritants including chafing behavior, whereby an organism curves their body to rub the convex portion of their body along a rough surface (Eibl-Eibesfeldt 1955; Wicklund 1969; Myrberg & Gruber 1974; Wyman & Walters-Wyman 1985; Mooring et al. 2004; Papastamatiou et al. 2007; Grossman et al. 2009; Ritter 2011; Berth et al. 2017).Dengue is one of the most prevalent infectious diseases around the world, present in all continents and mainly affecting developing countries. With few tools to fight and study this disease, it is imperative to have reliable animal models that not only recapitulate human disease but also contain human components to understand the pathogenic mechanism and immune responses, allowing the development of new treatments and vaccines against dengue. Humanized mice are a significant advance in the development of in vivo models to understanding the relation of the human immune system and target organs such as the liver during the infection by dengue virus, allowing basic and preclinical research. In this chapter, we describe the use of humanized NSG mice (huNSG) for the study of dengue disease. The first model describes reconstitution of the human immune system by transplanting human CD34+ stem cells in newborn or adult NSG mice. The second model combines the reconstitution with CD34+ stem cells with the transplant of human primary hepatocytes. This dual reconstituted animal will have two of the major players involved in the development of dengue infection. However, there are still more biological components missing in this model for dengue, but researchers continue working to improve the huNSG model to reconstitute other human components.The analysis of dengue virus (DENV) infected tissues in mice experimental model and in human biopsies/autopsies may support the pathogenesis studies. Through such models, it is possible to investigate possible histopathological changes caused by the infection and detections of different targets of interest, such as viral antigens, immune cells, and cytokines. In this chapter, we showed a brief review of how histological and immunohistochemistry approaches may improve the knowledge in this field.Dengue is an infectious disease caused by Dengue Virus, mainly transmitted by Aedes aegypti mosquitoes. Severe dengue is a potentially fatal syndrome in consequence of overwhelmed inflammation, in which thrombocytopenia and increased vascular permeability are frequently observed. Several experimental evidences point to the participation of both microvesicles (MVs) and circulating lipoproteins in inflammatory amplification in dengue pathogenesis. On this regard, many protocols for isolating plasma MVs have shown lipoproteins as the main contaminant. This is a limitation to studies aiming at the functional characterization of MVs, since both MVs and lipoproteins can modulate inflammatory responses. Here, we describe a biphasic density-based gradient ultracentrifugation as a tool for concomitant isolation of MVs and lipoproteins without cross-contamination. Erastin clinical trial Flow cytometry for MVs quantification and western blot for detection of apoB100 may be used to confirm the isolation and purity of the MVs.The Dengue pathophysiology has had several aspects determined over the years. However, some points remain elusive, such as the metabolic factors that regulate the massive B cell differentiation into antibody-secreting cells observed in Dengue patients. In this chapter, we describe an in vitro method capable of mimicking this Dengue-induced cell expansion. More specifically, this approach allows dengue virus-stimulated peripheral blood mononuclear cells (PBMCs) from healthy individuals to enhance the frequency of phenotypical and functional antibody-secreting cells (ASCs) after 7 days of culture. A manuscript recently published by Bonezi and colleagues displays results generated through this methodology.Despite many advances on the understanding of dengue pathogenesis in the last decades, some questions remained to be clarified. The virulence of the pathogen and the host immune response are the main factors involved in pathogenesis of dengue infection. In addition, skin dendritic cells (DCs) are one of the primary targets for dengue virus infection. After infection, DCs process and present antigens to T cells and also secrete cytokines that shape the immune response. Although relevant for the development of antiviral immune response, an imbalance in the cytokine production by immune cells could lead to cytokine storm observed in severe dengue fever cases. Therefore, this chapter will describe the protocols for the in vitro differentiation of human monocytes into human monocyte-derived dendritic cells (mdDCs), followed by dengue virus infection, as well as the cytokine quantification produced by mdDCs using a cytometric bead array method.This chapter will discuss reliable and relatively easy and fast strategies to evaluate the integrity of endothelial cell monolayers when infected by dengue virus (DENV). Human brain microvascular endothelial cells (HBMEC) were exploited here as general model of vessel wall core, but it may also be used as an in vitro simplified model of blood brain barrier (BBB). The integrity of endothelial cells monolayer can be inferred using a transwell culture system by (1) measuring transendothelial electrical resistance (TEER) using a Voltohmmeter; (2) analyzing the monolayer permeability to fluorescent-conjugated proteins and fluorimetric assay; (3) investigating virus extravasation by quantitative RT-PCR and plaque conventional assay. The rational to use those strategies is that vascular alterations are often observed during dengue infection, being associated to disease severity. The vasculature core consists of a barrier of endothelial cells, which are tightly adhered by the expression of adhesion molecules and tight junctions. This structure must be preserved in order to control the flux of cells and metabolites from the circulation to the tissues and to maintain vascular homeostasis. Therefore, experimental assays that allow evaluation of endothelial integrity can be useful platforms to further understand disease pathogenesis and screen pharmaceutical interventions to control vascular disturbance.A growing body of evidence demonstrates that endothelial cells (ECs) play a prominent role in immune-enhanced pathology seen in dengue virus (DENV) infection that might contribute to vascular permeability and hemorrhagic manifestations in severe dengue cases. However, it remains a question of whether DENV infection of ECs directly causes permeability or if extra-endothelial factors such as immune cell activation or antibody-dependent enhancement (ADE) are required. In this chapter, we detail the measurement of the transendothelial electrical resistance (TEER), a quantitative technique to measure the integrity of tight junction dynamics in cell culture models of endothelial monolayers and show that DENV infection of ECs does not cause endothelial permeability in vitro.A reliable and specific diagnosis is imperative in viral diagnosis, both for clinical management and surveillance, and to ensure that early treatment and control measures are carried out. The number of days of illness is important to choose the most appropriate method to be used and for the correct interpretation of the results obtained. Specific IgM is elicited after that period, indicating an active infection and usually lasts up to 3 months. However, in DENV secondary infections, IgM levels may be significantly lower or undetectable. After 10-12 days, a lifetime specific IgG is produced. link2 Routinely, the laboratory diagnosis of DENV infections can be performed by viral isolation and/or detection of viral nucleic acid, serological assays for the detection of specific antibodies (IgM/IgG), antigen (NS1) and the detection of viral antigens in tissues, which are suitable during certain phases of the disease. For serological diagnosis, serum, plasma, or cerebrospinal fluid (CSF) samples may be investigated. If the test is carried out a few days after collection, the specimens can be stored at 4 °C, since the immunoglobulins are stable in serum or plasma. If the storage period is extended, the material must be kept at -20 °C or -70 °C. In serology, several methods can be used to detect specific viral antigens and/or antibodies, produced by the host in response to DENV infection. Routinely, serological tests include the hemagglutination inhibition (HI) assay, the plaque reduction neutralizing test (PRNT), the gold standard assay for dengue immune response characterization, and ELISAs to detect IgM (MAC-ELISA) and IgG (IgG-ELISA).Several protocols for genomic amplification using reverse transcription followed by polymerase chain reaction (RT-PCR), important in the identification of the infecting serotype, have been used in the rapid diagnosis of Dengue Virus (DENV) infections. The qualitative protocol described by Lanciotti et al. (J Clin Microbiol 30 545-551, 1992) suggested by WHO detects the four DENV serotypes simultaneously in one procedure "semi-nested," generating amplified products with specific sizes in base pairs for each serotype and it has been the most used in the past two decades. link3 However, advances in molecular diagnosis have enabled the development of RT-PCR in real time (qRT-PCR) based on the use of dyes and probes (SYBR green and TaqMan), which is performed in a single step and is capable of providing quantitative data. In addition to quantification, the advantages of qRT-PCR over conventional RT-PCR include speed, greater sensitivity and specificity, and low rate of false positives. Several protocols for the diagnosis and/or quantification of DENV have already been described. Non-PCR-based methods such as reverse transcription loop-mediated isothermal amplification have shown high sensitivities and specificities. RT-PCR and qRT-PCR techniques can be performed using serum, plasma, infected cells, mosquitoes, fresh, and paraffin-embedded tissues. However, despite fast and accurate, they are limited to samples collected during the acute phase of infection (up to 7 days after the onset of symptoms) and require specialized equipment and trained staff.Viral proteins evolve to benefit the interaction with host proteins during the infection and replication processes. A comprehensive understanding of virus interactome with host proteins may thus lead to the identification of molecular targets for infection inhibition. We present a procedure for isolating and identifying the dengue virus interactome with human plasma proteins. It comprises the fractionation of human plasma by anion exchange chromatography, followed by affinity purification and mass spectrometry identification of the captured proteins. This procedure was applied to the characterization of the interactions of the four serotypes of dengue virus with human plasma proteins, mediated by the domain III of the envelope protein of the virus. The resulting interactome comprises 62 proteins, six of which were validated as new direct interactions of the virus with its human host.